Electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration

ABSTRACT

Disclosed are methods using neutral red to mediate the interconversion of chemical and electrical energy. Electrically reduced neutral red has been found to promote cell growth and formation of reduced products by reversibly increasing the ratio of the reduced:oxidized forms of NAD(H) or NADP(H). Electrically reduced neutral red is able to serve as the sole source of reducing power for microbial cell growth. Neutral red is also able to promote conversion of chemical energy to electrical energy by facilitating the transfer of electrons from microbial reducing power to a fuel cell cathode.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 09/793,025 filed Feb.26, 2001 which is a continuation-in-part of U.S. Ser. No. 09/350,072,filed Jul. 8, 1999, now U.S. Pat. No. 6,270,649, which claims priorityto U.S. Provisional Ser. No. 60/092,190 and Ser. No. 60/092,191, bothfiled Jul. 9, 1999. These applications are incorporated by referenceherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support in the form of theUnited States Department of Energy grant DE-FG02-93ER20108. The UnitedStates may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Microbial fermentation and biotransformation reactions are beingemployed with increasing frequency in the production of a number ofcommercially and industrially important products. There is also growinginterest in developing alternative energy sources through microbialfermentation of waste materials. The economic feasibility of theseprocesses depends on maximizing the efficiency of the fermentation orbiotransformation reactions.

Bacterial species are able to use various energy sources, includinglight and diverse organic and inorganic chemicals, for growth andmetabolism. These energy sources are used to produce an electrochemicalgradient that provides an electron donor for metabolism and allowsmaintenance of a membrane potential and proton motive force. Theenergetics of living systems are driven by electron transfer processesin which electrons are transferred from a substrate, which is therebyoxidized, to a final electron acceptor, which is thereby reduced.

In microbial metabolism, the energy produced from the driving force ofelectrons is directly proportional to the potential energy difference(ΔE₀′) between the initial electron donor (the first biochemicaldehydrogenating reaction) and final electron acceptor (e.g., the finalbiochemical hydrogenating reaction).

Certain microorganisms (e.g., Escherichia and Actinobacillus) are ableto grow using H₂ as an electron donor to reduce fumarate into succinatein an anaerobic respiration process. These bacteria obtain free energyand reducing power from the electron driving force generated by the E₀′difference between the coupled oxidoreduction half reactions of [2H⁺/H₂]and [fumarate/succinate].

Methanogens are strict anaerobic archea that can couple H₂ or HCOOHoxidation to CO₂ reduction into methane. Methanogenesis produces lessfree energy than other anaerobic respiration processes (e.g., fumarate,nitrate, or sulfate reduction) because the E₀′ difference between thehalf oxidation reduction reactions of [2H⁺/H₂] and [CO₂/CH₄] isrelatively small.

Hydrogen oxidation by microbial hydrogenases can be coupled to reductionof various biological electron carriers including NAD⁺, cytochromes, andquinones or to certain artificial redox dyes, such as methyl-viologenand neutral red (NR) (Annous, et al., 1996, Appl. Microbiol. Biotechnol.45:804-810, Kim, et al., 1992, J. Microbiol. Biotechnol. 2:248-254). Theeffect of redox dyes, with or without electrochemical reduction systems,on metabolite patterns and H₂ production has been examined in severalmicrobial processes, including the glutamate (Hongo, et al., 1979,Agric. Biol. Chem. 43:2083-2986), butanol (Girbal, et al., 1995,Microbiol. Rev. 16:151-162 and Kim, et al., 1992, J. Microbiol.Biotechnol, 2:268-272), and butyrate (Shen, et al., 1996, Appl.Microbiol. Biotechnol, 45:355-362) fermentations.

The specific activities of redox enzymes involved in bacterialcatabolism, such as hydrogenase or fumarate reductase, can be measuredusing their in vivo electron carriers (e.g., NAD or menanquinone) orwith artificial redox dyes (e.g., benzyl viologen) (Cecchini, et al.,1986, Proc. Natl. Acad. Sci. USA 83:8898-8902, Dickie, et al., 1979,Can. J. Biochem., 57:813-821, Kemner, et al., 1994, Arch. Microbiol.,161:47-54, Petrov, et al., 1989, Arch. Biochem. Bio-phys. 268:306-313,and Wissenbach, et al., 1990, Arch. Microbiol. 154:60-66). Bacteria thatproduce succinic acid as a major catabolic end product (e.g., E. coli,Wolinella succinogenes and other species) have a fumarate reductase(FRD) complex that catalyzes fumarate-dependent oxidation ofmenaquinone. This reaction is coupled to the generation of atransmembrane proton gradient that is used by the organism to supportgrowth and metabolic function (Kortner, et al., 1992, Mol. Microbiol.4:855-860 and Wissenbach, et al., 1992, Arch. Microbiol. 158:68-73). Thefumarate reductase of E. coli is composed of four nonidentical subunits:FRDA, FRDB, FRDC, and FRDD. The subunits are arranged in two domains:(i) the FRDAB catalytic domain and the FRDCD membrane anchor domain,which is essential for electron transfer and proton translocationreactions involving menaquinone (Cecchini, et al., 1995, J. Bacteriol.177:4587-4592, Dickie, et al., 1979, Can. J. Biochem., 57:813-821, andWestenberg, et al., 1990, J. Biol. Chem. 265:19560-19567). Subunits FRDAand FRDB retain catalytic activity in solubilized membrane preparations.

Electrochemical techniques employing redox dyes are useful forinvestigating the oxidation-reduction characteristics of biologicalsystems and provide information about biological energy metabolism(Moreno, et al., 1993, Eur. J. Biochem. 212:79-86 and Sucheta, et al.,1993, Biochemistry 32:5455-5465). Redox dyes that are useful inbioelectrochemical systems must easily react with both the electrode andthe biological electron carriers. Many biological electron carriers,such as NAD (Miyawaki, et al., 1992, Enzyme Microb. Technol. 14:474-478and Surya, et al., 1994, Bioelectrochem. Bioenerg. 33:71-73), c-typecytochromes (Xie, et al., 1992, Bioelectrochem. Bioenerg. 29:71-79),quinones (Sanchez, et al., 1995, Bioelectrochem. Bioenerg. 36:67-71),and redox enzymes, such as nitrite reductase (White, et al., 1987,Bioelectro-chem. Bioenerg. 26:173-179), nitrate reductase (Willner, etal., 1992, Bioelectrochem. Bioenerg. 29:29-45), firnarate reductase(Sucheta, et al., 1993, Biochemistry. 32:5455-5465), glucose-6-phosphatedehydrogenase (Miyawaki, et al., 1992, Enzyme Microb. Technol.14:474-478), ferredoxin-NADP reductase (Kim, et al., 1992, J. Microbiol.Biotechnol. 2:2771-2776) and hydrogenase (Schlereth, et al., 1992,Bioelectrochem. Bioenerg. 28:473-482) react electrochemically with theredox dyes.

Certain redox dyes with lower redox potentials than that of NAD, such asmethyl viologen (MV) (Kim, et al., 1988, Biotechnol. Lett. 10:123-128,Pequin, et al., 1994, Biotechnol. Lett. 16:269-274, and White, et al.,1987, FEMS Microbiol. Lett. 43:173-176), benzyl viologen (Emde, et al.,1990, Appl. Environ. Microbiol. 56:2771-2776), and neutral red (NR)(Girbal, et al., 1995, FEMS Microbiol. Rev. 16:151-162 and Kim, et al.,J. Biotechnol. 59:213-220) have been correlated with alterations in therate of biological redox reactions in vivo. Hongo and Iwahara (Hongo, etal., 1979, Agric. Biol. Chem. 43A:2075-2081 and Hongo, et al., 1979,Agric. Biol. Chem. 43B:2083-2086) discovered that including redox dyeswith low ΔE₀′ values (e.g., MV, benzyl viologen and NR) in bacterialfermentation conducted under cathodic reduction conditions wascorrelated with an increase in L-glutamate yield (about 6%). In themethod of Hongo and Iwahara, a platinum electrode was used to deliverelectricity at a level that was sufficiently high to generate hydrogenfrom water. Therefore, the source of increased reducing power in themethod of Hongo and Iwahara is not known, nor was the mechanism by whichthe tested dyes affect fermentation characterized. Addition of NR toacetone-butanol fermentations is correlated with decreased production ofacids and H₂, and enhanced production of solvent (Girbal, et al., 1995,FEMS Microbiol. Rev. 16:151-162 and Kim, et al., 1992, J. Microbiol.Biotechnol. 2:2771-2776), an effect that was further enhanced underelectroenergized fermentation conditions (Ghosh, et al., 1987, abstr.79. In Abstracts of Papers, 194th ACS National Meeting. AmericanChemical Society). Viologen dyes have been used as electron mediatorsfor many electrochemical catalytic systems using oxidoreductases invitro and in vivo (James, et al., 1988, Electrochem. Bioenerg. 20:21-32,Kim, et al., 1988, Biotechnol. Lett. 10:123-128, Moreno, et al., 1993,Eur. J. Biochem. 212:79-86, Schlereth, et al., 1992, Bioelectrochem.Bioenerg. 28:473-482, and White, et al., 1987, FEMS Microbiol. Lett.43:173-173).

An electrochemical system was used to regenerate reduced iron for growthof Thiobacillus ferrooxidans on electrical reducing power (Robinson, etal., 1982, Can. J. Biochem. 60:811-816).

It may be possible to control or alter metabolism by linking biochemicalprocesses to an external electrochemical system. Linking biochemical andelectrochemical systems may allow the use of electricity as a source ofelectrons for bacterial growth and in vivo or in vitro fermentation orbiotransformation reactions.

A reversible biochemical-electrochemical link may allow conversion ofmicrobial metabolic or enzyme catalytic energy into electricity. Biofuelcells in which microbial energy is directly converted to electricalenergy using conventional electrochemical technology have been described(Roller, et al., 1984, J. Chem. Tech. Biotechnol. 34B:3-12 and Allen, etal, 1993, Appl. Biochem. Biotechnol. 39-40:27-40). Chemical energy canbe converted to electric energy by coupling the biocatalytic oxidationof organic or inorganic compounds to the chemical reduction of theoxidant at the interface between the anode and cathode (Willner, et al.,1998, Bioelectrochem. Bioenerg. 44:209-214). However, direct electrontransfer from microbial cells to electrodes has been shown to take placeonly at very low efficiency (Allen, et al., 1972, J. R. Norris and D. W.Ribbons (eds.). Academic Press, New York, 6B:247-283).

The electron transfer efficiency can be improved by using suitable redoxmediators (Bennetto, et al., 1985, Biotechnol. Lett. 7:699-105), andmost of the microbial fuel cells studied employed electron mediatorssuch as the redox dye thionin (Thurston, et al., 1985, J. Gen.Microbiol. 131:1393-1401). In microbial fuel cells, two redox couplesare required for: (1) coupling the reduction of an electron mediator tobacterial oxidative metabolism; and (2) coupling the oxidation of theelectron mediator to the reduction of the electron acceptor on thecathode surface (where the electron acceptor is regenerated byatmospheric oxygen) (Ardeleanu, et al., 1983, Bioelectrochem. Bioenerg.11:273-277 and Dealney, et al., 1984, Chem. Tech. Biotechnol.34B:13-27).

The free energy produced by either normal microbial metabolism or bymicrobial fuel cell systems is mainly determined by the potentialdifference (ΔE₀′) between the electron donor and acceptor according tothe equation, −ΔG=nFΔE₀ in which a G is the variation in free energy, nis the number of electron moles, and F is the Faraday constant (96,487J/volt) (Dealney, et al., 1984, Chem. Tech. Biotechnol. 34B:13-27).Coupling of the metabolic oxidation of the primary electron donor (NADH)to the reduction of the final electron acceptor (such as oxygen orfumarate in bacterial respiration systems) is very similar to thecoupling of electrochemical half-reaction of the reductant (electrondonor) to the half reaction of the oxidant (electron acceptor) in a fuelcell or battery system (Chang, et al., 1981, 2nd ed., MacmillanPublishing. New York). Biological reducing power sources such as NADH(E₀′=−0.32 volt), FdH₂ (E₀′=−0.42 volt), or FADH₂ (E₀′=−0.19 volt) withlow redox potentials can act as reductants for fuel cells, but they arenot easily converted to electricity because the cytoplasmic membranemust be non-conductive to maintain the membrane potential absolutelyrequired for free energy (i.e., ATP) production (Thauer, et al., 1997,Bacteriol. Rev. 41:100-180).

For electron transfer to occur from a microbial electron carrier to anelectrode, an electron mediator is required (Fultz, et al., 1982, Anal.Chim. Acta. 140:1-18). Allen, et al. (1993, Appl. Biochem. Biotechnol.39-40:27-40) reported that the reducing power metabolically produced byProteus vulgaris or E. coli can be converted to electricity by usingelectron mediators such as thionin. Tanaka, et al. (1985, Chem. Tech.Biotechnol. 35B:191-197 and 1988, Chem. Tech. Biotechnol. 42:235-240)reported that light energy can be converted to electricity by Anabaenavariabilis using HNQ as the electron mediator. Park, et al. (1997,Biotech. Techniq. 11:145-148) confirmed that viologen dye cross-linkedwith carbon polymers and adsorbed to Desulfovibro desulfuricanscytoplasmic membranes can mediate electron transfer from bacterial cellsto electrodes or from electrodes to bacterial cells.

There remains a need in the art for improved, more efficient methods forconverting metabolic reducing power to electrical energy, and forconverting electrical energy to metabolic reducing power.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention is a method of promoting reductiveprocesses in a bioreactor system comprising the steps of (a) providingan electrochemical bioreactor system having a cathode compartmentequipped with a cathode and an anode compartment equipped with an anode,the cathode and anode compartment being separated by a cation selectivemembrane, wherein the cathode and anode are connected by a conductivematerial to a power supply; (b) placing a suitable amount of neutral redand a biological catalyst in the cathode compartment.

In a particularly advantageous form of the invention, the biologicalcatalyst is an enzyme that uses NADH or NADPH as a cofactor. The cathodecompartment comprises NADH or NADPH and an oxidized substrate for theenzyme. Electrically reduced neutral red transfers electrons to NAD⁺ orNADP⁺. In a preferred form of the invention, the enzyme isoxidoreductase, most particularly in alcohol dehydrogenase, the oxidizedsubstrate is an aldehyde or ketone and the reduced product is analcohol.

Another aspect of the invention is a method for generating electricityusing a biological system comprising the steps of (a) providing anelectrochemical fuel cell system comprising an anode compartment and acathode compartment separated by a cation-selective membrane, whereineach compartment is equipped with an electrode, wherein the electrodesare connected by a wire to a multimeter; (b) placing an anolyte in theanode compartment, the anolyte comprising a suitable concentration ofneutral red and a biological catalyst selected from the group consistingof bacteria, archea, plant cells, and animal cells; (c) placing asuitable catholyte in the cathode compartment; and (d) allowing theneutral red-mediated conversion of chemical reducing power toelectricity.

It is an object of the invention to provide methods that allow theinterconversion of biochemical reducing power (e.g., NADH), biologicalenergy (ATP), and electrical energy in an electrochemical bioreactor orfuel cell.

It is a further object of the invention to provide an economical methodof promoting cell growth or production of desired products usingelectrically reduced neutral red.

Another object of the invention is to provide a method for convertingbiological reducing power into electricity.

It is an advantage of the present invention that electrical energy maybe used to promote cell growth or fermentation or enzymatictransformation in the presence of neutral red.

Another advantage of the invention is that neutral red promotes thegeneration of electrical energy from waste material comprising mixedbacterial populations.

Other objects, features, and advantages of the present invention will beapparent on review of the specification and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of a microbial fuel cell using neutral red(NR) as an electronophore.

FIG. 2 shows the current production from NADH oxidation in a chemicalfuel cell with NR (A) or thionin (B) as the electron mediator.

FIG. 3 is a cyclic voltammogram obtained with a glassy carbon electrodeon successive cycles following introduction of the electrode into a 100μM NAD⁺ solution.

FIG. 4 shows the current and potential obtained in a glucose fuel cellusing E. coli K-12 resting cells and neutral red or thionin.

FIG. 5 shows the electrical current and potential levels obtained usingA. succinogenes growing or resting cells.

FIG. 6 shows the current and potential produced in a glucose (3 g/L)fuel cell using anaerobic sewage sludge as catalyst and NR (100 μM) asthe electronophore.

FIG. 7 is a proposed model of the energy flow in cells under normal (A)or electrogenic (B) glucose metabolism.

FIG. 8 is a time course for biotransformation of β-tetralone toβ-tetralol by the yeast T. capitacum at 1 g/L of substrate in thepresence and absence of 1.5 volt electricity.

FIG. 9 is a time course of biotransformation of β-tetralone toβ-tetralol by the yeast T. capitacum at 2 g/L of substrate in thepresence and absence of 1.5 volt electricity.

FIG. 10 is the effect of pulse feeding of 2 g/L of substrate on thebiotransformation of β-tetralone to β-tetralol.

FIG. 11 is the effect of pulse feeding of 1 g/L of substrate on thebiotransformation of β-tetralone to β-tetralol.

FIG. 12 is the effect of ethanol concentration on the biotransformationof β-tetralone to β-tetralol.

FIG. 13 is the effect of electrical potential on the biotransformationof β-tetralone to β-tetralol.

FIG. 14 is the effect of electrical potential on the biotransformationof β-tetralone to β-tetralol.

FIG. 15 is the biotransformation kinetics with purified β-tetralonereductase with NAD in the presence of electricity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for achieving the efficientinterconversion of chemical and electrical energy using neutral red. Oneaspect of the invention is a method for using electrical energy as asource of reducing power in fermentation or enzymatic reactions. Anotheraspect of the invention includes a method of using neutral red and cellsor enzymes to produce electricity.

The invention is based on the discovery that the use of neutral red inmethods directed toward regulating electron flow in biological systemsoffers a number of surprising advantages, which are disclosed in relatedU.S. Ser. Nos. 60/092,190 and 60/092,191; Park and Zeikus, J. Bacteriol.181:2403-2410, 1999; and Park, et al. Appl. Environ. Microbiol. Inpress, all of which are incorporated by reference in their entirety.

A critical factor for the control of end-product yields in fermentationor enzymatic biotransformation reactions is regulation of electrondistribution through the NADH/NAD⁺ ratio. If additional reducing power(e.g., H₂ or electrochemically produced reducing equivalents) issupplied to bacteria, an increase in the NADH/NAD⁺ ratio and metabolismmay be expected. However, efficient transfer of electrons fromelectricity to NAD⁺ requires a suitable electron mediator.

As discussed in detail in U.S. Ser. Nos. 60/092,190 and 60/092,191,neutral red was discovered to be a particularly good electron mediatorfor use in the interconversion of electricity and metabolic reducingpower in electrochemical bioreactor systems. Neutral red is able to forma reversible redox couple at the electrode and has a highly negativeE₀′. The E₀′ value for neutral red is very similar to that ofphysiological electron carriers in the electron transport chain,including, for example, NADH. The ability of neutral red to acceptelectrons from far up the electron transport chain enhances electricityproduction in biofuel cell systems. Neutral red is soluble at a neutralpH, it is stable in both its oxidized and reduced forms, it does notdecompose during long-term redox cycling.

As disclosed in U.S. Ser. Nos. 60/092,190 and 60/092,191, neutral red isrelatively nontoxic, and can be easily adsorbed on the cytoplasmicmembrane of the cells under study, where it functions as anelectronophore, or electron shuttle, for electron transfer across thecytoplasmic membrane. Neutral red was demonstrated to function as anelectron mediator in reversible oxidation or reduction of compounds andto substitute for menaquinone in the cell membrane. Surprisingly,electrically reduced neutral red promotes growth, proton translocationand metabolite production in cells even in the absence of other sourcesof reducing power.

One aspect of the present invention provides a method for promotingreductive processes in a bioreactor system comprising the steps of (a)providing an electrochemical bioreactor system having a cathodecompartment equipped with a cathode and an anode compartment equippedwith an anode, the cathode and anode compartment being separated by acation selective membrane, wherein the cathode and anode are connectedby a conductive material to a power supply; and (b) placing a suitableamount of neutral red and a biological catalyst in the cathodecompartment.

Preferably, the biological catalyst is selected from the groupconsisting of microbial cells, plant cells, animal cells, isolatedintact cytoplasmic membranes, solubilized cytoplasmic membranes, and anenzyme having NADH or NADPH a cofactor. To maximize the efficiency ofthe interconversion of biochemical and electrical energy, the biologicalcatalyst is immobilized on the cathode.

In a preferred embodiment, the method of the invention further comprisesthe steps of (c) placing an anolyte solution in the anode compartment;(d) delivering to the cathode an electric current of suitable strengthto cause reduction of at least a portion of oxidized neutral red in thecathode compartment; and (e) allowing the reduced neutral red totransfer electrons to an oxidized substrate or an electron carrier.

The method of the invention is very versatile, in that it can be adaptedfor use with any number of biological catalysts, including microbial,plant, or animal cells, isolated intact cell membranes, solubilizedcytoplasmic membranes, or a preparation of an enzyme that uses NADH orNADPH as a cofactor. Most conveniently, the biological catalystcomprises substantially pure or mixed cultures of cells, or an enzymepreparation. Preferably, the biological catalyst is capable of promotingthe reduction of an oxidized substrate to a commercially or industriallyimportant product, such as succinate, methane, or alcohols.

When whole cells are used as the biocatalyst, electrically reducedneutral red promotes cell growth or formation of a reduced product bychemical reduction of an NAD⁺ or NADP⁺ cofactor, or by serving as anelectronophore. Preferably, the bioreactor system is one in which theelectrically reduced neutral red promotes cell growth, ATP synthesis, orformation of a reduced product by chemical reduction of an NAD⁺ or NADP⁺cofactor or by functioning as an electronophore.

In the examples below, electrically reduced neutral red is shown topromote the reduction of fumarate to form succinic acid in fermentationreactions using Actinobacillus succinogenes in a bioreactor system.Because succinic acid is an important fermentation product having manyindustrial uses, there is interest in developing a more efficientfermentation process with enhanced succinic acid yields.

It was discovered that including electrically reduced neutral red duringgrowth of A. succinogenes on glucose medium in a bioreactor systempromotes fumarate reduction by chemically reducing NAD⁺. Furthermore,neutral red promotes succinic acid production through its function as anelectron mediator and electronophore. The electrical reduction ofneutral red (E₀′=−0.325 volt) is chemically linked to NAD⁺ reduction,and it is biochemically linked to generation of a proton motive forceand succinate production. Neutral red appears to function by replacingmenaquinone (E₀′=−0.073 volt) in the membrane bound fumarate reductasecomplex. Preferably, the reduced neutral red is able to increase cellgrowth by at least 10%, 20%, or even as much as 40% or more, relative toa comparable bioreactor system lacking neutral red. Electrically reducedneutral red is able to increase glucose or fumarate consumption by atleast 25%, 50%, or 100% or more. Succinate production is increased byabout 10% or even as much as 25% or more, relative to the productionlevels observed in a comparable bioreactor system lacking neutral red.

Similarly, electrically reduced neutral red is able to substitute for H₂in promoting the growth of methanogenic bacteria and the reduction ofCO₂ to methane by methanogenic archea. Preferably, the method of theinvention increases growth of archea or methane production by at leastabout 25%, 50%, 100% or even as much as 300% or more.

It is reasonable to expect that the method of the present invention maybe used with a wide range of biocatalysts to promote cell growth or theformation of reduced products in electrochemical bioreactor systems. Itis envisioned that the method can be used with a variety of bacteria,archea, plant cells or animal cells.

It is expected that enzyme preparations may also be used in the practiceof the invention. A desired enzyme may be partially purified usingstandard methods known to one of ordinary skill in the art. The enzymemay be isolated from its native source or from a transgenic expressionhost, or obtained through a commercial vendor.

Useful enzymes include any enzyme that can use reducing power fromelectrically reduced neutral red to form a desired reduced product, orwhich can transfer reducing power to neutral red and form a desiredoxidized product. Most commonly, this reduction is mediated by NADPH orNADH. It is reasonably expected that any oxidoreductase may be used inthe practice of the invention. For example, isolated alcoholdehydrogenases could be used in a bioreactor system comprisingelectrically reduced neutral red, NADP⁺ or NAD⁺, and a ketone, aldehydeor carboxylic acid that can serve as a substrate for the enzyme to forma more reduced end product such as an alcohol. Another example of auseful enzyme is carboxylic acid reductase, which uses NADPH and ATP toconvert a carboxylic acid to reduced products (U.S. Pat. No. 5,795,759,herein incorporated by reference). One skilled in the art wouldappreciate that most enzyme-catalyzed reactions are reversible, and thatthere may be applications in which one would wish to use anoxidoreductase to obtain a desired oxidized substrate by the method ofthe present invention.

In the electrochemical bioreactor used in the present invention, thebiocatalyst and neutral red are preferably immobilized on the cathode.In the case of whole cell biocatalysts, self-immobilization on a finewoven graphite felt electrode was found to take place. Immobilization ofthe biocatalyst may be achieved using any suitable method. Numeroustechniques for immobilizing biocatalysts are known to the art (forexample, see Woodward and Spokane, Analytical Enzymes: Biosensors inIndustrial Enzymology, 2d Edition, p. 51-59, incorporated by referenceherein). One wishing to immobilize a biocatalyst in the practice of thepresent invention could do so placing the biocatalyst, neutral red, andpyridine nucleotide cofactor between an electrode and an outer membrane(e.g., a polymer membrane) such that the biocatalyst, cofactor, andneutral red are sandwiched between the electrode and membrane.Alternatively, biocatalyst, neutral red, and pyridine nucleotidecofactor could be embedded in a matrix polymer and coated onto theelectrode.

One of ordinary skill in the art wishing to practice the presentinvention could readily prepare an electrochemical bioreactor or fuelcell using the teachings disclosed herein. It should be appreciated thatcertain modifications to the disclosed bioreactors and fuel cells arewell within the ability of one skilled in the art.

Catholytes and anolytes that may be used in electrochemical bioreactorsor in fuel cells are provided in the examples. Catholytes that have beenfound to be suitable in electrochemical bioreactors include bacterialgrowth media or a phosphate buffer (50-100 mM, pH 7.0-7.2). Othersuitable catholyte buffers for used in an electrochemical bioreactorinclude any catholyte that is non-denaturing to cells or enzymes.

A phosphate buffer comprising saline has been found to be suitable foruse in an electrochemical bioreactor (100 mM sodium phosphate (pH 6.0)and 100 mM NaCl. A suitable anolyte may include any anolyte that isnondenaturing to cells or enzymes.

For a fuel cell, neutral red (100 μM) and a bacterial cell suspension in50 mM phosphate buffer (pH 7.0) was found to be a suitable anolyte, with100 mM phosphate buffer (pH 7.0) and 50 mM ferricyanide as thecatholyte.

In both the electrochemical bioreactor systems and the fuel cell systemdescribed in the examples, the cathodic and anodic compartments wereseparated by a Nafion cationic selective membrane septum that allows thepassage of protons and cations only. A suitable membrane for separatingthe cathodic and anodic compartments can be any membrane that allowstransfer of only protons or cations across the membrane.

In the electrochemical bioreactor systems described in the examplesbelow, the electrodes were made from fine woven graphite felt. The wovengraphite felt offers the advantage of providing a large surface areaelectrode that permits immobilization of the biocatalyst over a largearea. However, other materials may be suitable for electrodes, includingconductive polymers and metallic materials.

The electrodes were connected to a power source or to a multimeter usinga platinum wire. Other materials suitable for connecting the electrodesto the power source or multimeter include conducting poolymers ormetallic materials.

In the electrical bioreactors described below, the current between theanode and cathode was between about 0.4 and about 2.0 mA, with thevoltage being about 1.5 V. It is envisioned the present invention couldbe practiced using currents of from about 0.004 to about 200 mA.

In the fuel cell system, the resistance from the anode and cathode wasabout 1,000 ohms. It is envisioned that resistances of from about 10 toabout 10,000 ohms could be used in the practice of the invention.

Neutral red was included in the catholyte of electrochemical bioreactorsand in the anolyte of fuel cell systems at a concentration of about 100μM. It is expected that neutral red concentrations of between about 1and 1000 μM would be suitable in the practice of the invention.

Neutral red can also be used as an electron mediator in the conversionof energy derived from the metabolism of growing or resting bacterialcells to electricity.

Using Actinobacillus succinogenes 130Z growing cells in a fuel cellsystem that had neutral red as the electron mediator and ferricyanide asthe electron acceptor, the maximum current produced using was 2.17 mA,and the potential was <100 mV in a closed circuit configuration. After20 hour cultivation, the fuel cell system was converted from a closed toan open circuit system. The potential rapidly reached the theoreticalmaximum value of 0.685 volt (i.e. the redox potential difference betweenNR).

A comparison of the efficacy of NR and thionin as electron mediatorsmade using A. succinogenes resting cells as the catalyst revealed thatmuch more electricity was produced with NR than with thionin as theelectron mediator. When NADH, NR, and ferricyanide were used as theelectron donor, electron mediator, and electron acceptor, respectively,the current produced was proportional to NADH concentration. In a systemthat employed E. coli K-12 as the catalyst, the currents and voltagesproduced were similar to those obtained using A. succinogenes as thecatalyst. The current and voltage were found to increase with increasingglucose concentrations.

Anaerobic sewage sludge was also used as the catalyst in a fuel cellsystem. The voltage and current produced in fuel cells using sewagesludge as the catalyst were comparable to those produced using E. coliand Actinobacillus, and they were stable for 120 hours in a closedcircuit system with a 2.2 K ohms external resistance.

It is expected that growing or resting cells of types other than thosedescribed in the examples can be used as catalysts in a fuel cell systemto generate electricity by the method of the present invention.Depending on the particular cell chosen as a biocatalyst, reducing powerused in the generation of electricity may include light, inorganiccompounds, or organic compounds, or any other energy source that cellsare able to use for growth or metabolism.

It is envisioned that the neutral red-mediated interconversion ofbiochemical and electrical energy may be adapted for use in a number ofdifferent applications. For example, neutral red oxidoreduction can beused to detect electrical levels in biosensor systems using whole cellsor enzymes.

Accordingly, the invention includes a method for detecting the presenceof a specific organic or inorganic test compound in a sample comprisingthe steps of (a) providing biosensor comprising an electrochemical fuelcell system having an anode compartment and a cathode compartmentseparated by a cation-selective membrane, wherein each compartment isequipped with an electrode, wherein the electrodes are connected by awire to a multimeter; (b) placing an anolyte in the anode compartment,the anolyte comprising the sample, a suitable concentration of neutralred, and a biological catalyst comprising microbial cells and an enzyme,wherein the biological catalyst is able to oxidize the test compound;(c) placing a suitable catholyte in the cathode compartment; and (d)allowing oxidation of at least a portion of any test compound present inthe sample and reduction of at least a portion of oxidized neutral red;(e) allowing the transfer of electrons from reduced neutral red to thecathode; (f) detecting the generation of an electrical current.

In cell or enzyme biosensors known to the art, the presence of achemical (e.g., glucose) is detected using an enzyme (glcuose oxidase)in a membrane-based electrode system. In the example of glucose andglucose oxidase, the enzyme-catalyzed reaction consumes O₂ and producesperoxide. Therefore, glucose present in the sample is correlated with adecrease in O₂ concentration and an increase in peroxide concentration,either one of which be detected by a specific electrode. By the methodof the present invention, electrical current generated can be measureddirectly. In the neutral red system, a specific compound in an unknowntest sample is tested using cells or enzymes that are capable ofoxidizing the compound to generate a detectable current upon oxidationof the compound by the biocatalyst. Therefore, the concentration of thecompound can be determined by measuring the electricity generated uponoxidation of the test compound. It is well within the ability of oneskilled in the art wishing to detect a particular compound to adapt themethod of the present invention to detect the compound by selecting asuitable biocatalyst capable of oxidizing the compound.

Another important application using neutral red provides a method formeasuring the chemical oxygen demand in waste water comprising (a)providing an electrochemical fuel cell system comprising an anodecompartment and a cathode compartment separated by a cation-selectivemembrane, wherein each compartment is equipped with an electrode,wherein the electrodes are connected by a wire to a multimeter; (b)placing an anolyte in the anode compartment, the anolyte comprising asuitable concentration of neutral red and waste water comprising orsupplemented with a biological catalyst; (c) placing a suitablecatholyte in the cathode compartment; (d) allowing the neutralred-mediated conversion of chemical reducing power to electricity; (e)measuring the electrical current generated by the fuel cell system.

The following nonlimiting examples are intended to be purelyillustrative.

EXAMPLES Example 1 Electrically Reduced Neutral Red Promotes theReduction of Fumerate to Succinic Acid

Chemicals and Reproducibility of Results

All chemicals were reagent grade and gases were purchased from AGAChemicals (Cleveland, Ohio, USA). All individual experiments wererepeated two to three times with identical results.

Electrochemical Bioreactor Systems

The ECB system I (40 ml working volume) was used for enzymatic andchemical reduction tests and ECB system II (300 ml working volume) wasused for electrical-dependent cultivation of cells. The ECB systems,specially designed for maintaining anaerobic conditions and for growingbacteria, were made from Pyrex glass by the MSU Chemistry Department,East Lansing, Mich., USA. The ECB system was separated into anode andcathode compartments by a cation selective membrane septum (diameter[φ]=22 mm for type I and [φ]=64 mm for type II) (Nafion,Electrosynthesis, Lamcosta, N.Y.); 3.5 Ωcm⁻² in 0.25 N NaOH). Chemicalsand metabolites cannot be transferred across the Nafion membrane; onlyprotons or cations transfer. Both the anode and cathode were made fromgraphite fine woven felt (6 mm thickness, 0.47 m²g⁻¹ available surfacearea (Electrosynthesis, N.Y., USA). A platinum wire ([φ] Ω0.5 mm, <1.0Ωcm⁻²; Sigma, St. Louis, Mo., USA) was attached to the graphite feltusing graphite epoxy (<1.0 Ωcm⁻², Electrosynthesis, N.Y., USA). Theelectric resistance between anode and cathode was <1 kΩ. The weight ofboth electrodes was adjusted to 0.4 g (surface area, 0.188 m²) forsystem 1 and 3.0 g (surface area, 1.41 m²) for system II. The currentand voltage between anode and cathode were measured by precisionmultimeter (Fluke model 45, Everett, Wash., USA) and adjusted to 0.3-2.0mA and 1.5 volt for system I, and 1.0-10.0 mA and 2.0 volt for systemII, respectively. The electrochemical half oxidation of H₂O was coupledto half reduction of NR (100 μM) and the oxidation of reduced NR wascoupled to bacteriological reduction of fumarate. H₂ was not producedunder the electrochemical conditions used to reduce NR or MV. For testsin ECB system I, the cathode compartment contained the cell suspension,membrane suspension or solubilized membranes and the anode compartmentcontained 50 mM phosphate buffer (pH 7.2) and 100 mM NaCl. For growthstudies in ECB system II, the cathode compartment contained the growthmedium inoculated with A. succinogenes and the anode compartmentcontained 100 mM phosphate buffer (pH 7.0) and 100 mM NaCl.

Organism and Growth Conditions

A. succinogenes type strain 130Z is maintained at MBI International(Lansing, Mich., USA) (10, 39). Bacteria were grown inbutyl-rubber-stoppered, 158 ml serum vials containing 50 ml medium withCO₂—N2 (20%-80%, 20 psi) gas phase, unless stated otherwise. The growthmedium A contained the following (per liter of double distilled water):yeast extract, 5.0 g; NaHCO₃, 10.0 g; NaH₂PO₄.H₂O, 8.5 g; and Na₂HPO₄,12.5 g. The pH of medium was adjusted to be 7.0 after autoclaving.Separately autoclaved solutions of glucose (final concentration 60 mM),and fumarate (final concentration 50 mM) were aseptically added to themedium after autoclaving. Media were inoculated with 5.0% (v/v) samplesof cultures grown in the same medium and incubated at 37° C.

Preparation of Cell Suspensions

Bacterial cultivation, harvest and washing were done under strictanaerobic N₂ atmosphere as described previously (39). A 16 hour A.succinogenes culture was harvested by centrifugation (5,000×g, 30minutes) at 4° C. and washed three times using a 1500 ml solution of 50mM Na phosphate buffer (pH 7.2) containing 1 mM dithiothreitol (DTT).The washed bacterial cells were re-suspended in 50 mM sodium phosphatebuffer with 2 mM DTT. This suspension was used as a catalyst forH₂-dependent and electrical-dependent reduction of fumarate tosuccinate; and, it was used for cyclic voltammetry and for NR absorptionto cells.

Electrochemical Reduction of NAD⁺ or NADP⁺

ECB system I with 1 mM NAD⁺ or NADP⁺ and 100 (ΩM NR or MV was used forelectrochemical reduction of NAD⁺ or NADP⁺. The electrode potential andcurrent were adjusted to 2.0 volts and, 1.0-3.0 mA, respectively.Ag/AgCl and platinum electrodes were used to measure the reactants redoxpotential to check if the reaction was progressing. Generally, the redoxpotential of a biochemical or electrochemical reaction is measured usingan Ag/AgCl electrode (E₀′ of [Ag/Ag⁺], =+0.196 volt) or a Calomelelectrode (E₀′ of [Hg/Hg⁺], +0.244) as a reference electrode but it hasto be expressed as the potential vs. natural hydrogen electrode (NHE),which is used for thermodynamical calculation of organic or inorganiccompounds (e.g., E₀′ of NADH/NAD⁺ is a −0.32 volt and H₂/2H+ is −0.42volt). A potential measured using Ag/AgCl electrode is converted topotential vs. NHE by adding +0.196 volt to the measured potential (E₀′vs. NHE=E₀′vs. Ag/AgCl+0.196). Oxygen was purged from the reactants andfrom the redox dye solution in 50 mM Tris-HCl (pH 7.5) by bubbling withoxygen free nitrogen for 10 minutes before supplying electricity. TheNADH concentration in the reactant was spectrophotometrically measuredat 340 mm and calculated using the millimolar extinction coefficient6.23 mM⁻¹ cm⁻¹. NAIDH or NADPH production was confirmed by absorptionspectra data at each sampling time.

Preparation of Purified Membranes Solubilized Membranes and MembraneFree Cell Extract

Cell free extracts were prepared at 4° C. under an anaerobic N₂atmosphere, as described previously (Van der Werf, et al., 1997, Arch.Microbiol. 167:332-342). The harvested and washed cells were resuspendedin 50 mM phosphate buffer (pH 7.2) containing 1 mM DTT and 0.05 mg/mldeoxyribonuclease. Cells were disrupted by passing twice through aFrench Press at 20,000 psi. The cell debris was removed bycentrifugation three times at 40,600×g for 30 minutes. The purifiedmembranes were obtained from the cell free extracts by centrifugation at100,000×g for 90 minutes. The supernatant was decanted and saved as themembrane-free cell extract. The brown and clear precipitate was washedtwice with 50 mM phosphate buffer (pH 7.2) and re-suspended in the samebuffer by homogenization. Solubilized membranes were obtained frommembrane fraction by Triton X-100 extraction (Lemire, et al, 1983, J.Bacteriol. 155:391-397). Triton X-100 was added to a final 1% (v/v)concentration and, the suspension was incubated for 3 hours.Triton-solubilized protein was recovered after removing insoluble debrisby centrifugation at 100,000×g and 4° C. for 90 minutes.

Neutral Red Binding to Cells and Membranes

The absorption of redox dyes to cells and purified membranes wasdetermined by measuring the residual NR and MV in solution after mixingwith cells or membrane suspensions for 30 minutes at 37° C. Bacterialcell suspensions (OD₆₆₀ between 0-3.0) and the purified membranesuspension (0-10 mg/ml protein) were used to analyze redox dyeabsorption (i.e., binding). NR solutions (50 μM and 25 μM) and MV (100μM) were used for measuring dye binding to intact cells and membranes.MV (100 μM) was used for cell binding. The cells and membranes wereremoved from the reaction mixture by centrifugation at 12,000×g for 10minutes and by ultracentrifugation at 150,000×g for 20 minutes,respectively. The NR concentration was calculated using a calibrationcurve spectrophotometrically pre-determined at 400 nm and pH 7.2, and MVwas determined using the millimolar extinction coefficient (578) 9.78mM⁻¹ cm⁻¹ after reduction by addition of Elepsiden 1.5 mM dithionite atpH 7.2 (Lissolo, et al., 1984, J. Biol. Chem. 259:11725-11729). Theprotein concentration of membrane suspensions was determined by acalibration curve (protein concentration, mg/ml=A₅₉₅×1.3327) usingBradford Reagent (Bio-Rad, Hercules, Calif., USA).

Measurement of Proton Translocation

Proton translocation was measured under an anoxic N₂ atmosphere.H₂-dependent proton translocation by cell suspensions was measured asdescribed by Fitz and Cypionka (Fitz, et al., 1989, Arch. Microbiol.152:369-376). Electrical-dependent proton translocation was measured inan electrochemical bioreactor system designed for measurement of protontranslocation. The tube ([φ] 10 mm ID and 90 mm length) with a Vycor tip(ion exchangeable hard membrane, Bas, West Lafayette, Ind., USA) wasused as an anode compartment and a graphite rod ([φ] 7 mm×70 mm) wasused as an anode, and 0.05 g graphite felt (surface area, 0.0235 m²) wasused as a cathode. The pH electrode (Orion 8103 ROSS) was placed in thecathode compartment and was connected to a recorder (Linear) via a pHmeter (Corning, 130) that converted the proton pulse into a recordablesignal. Cell suspensions were made in KKG solution (pH 7.1) whichcontains 100 mM KSCN, 150 mM KCl and 1.5 mM glycylglycin and placed inthe cathode. The anode contained a 50 mM phosphate buffer with 50 mM KClas an anolyte. The total volume and working volume of the cathode andanode compartments were 30 ml and 5.5 ml, respectively. The workingpotential and current between anode and cathode were 2.0 volt and0.3-0.35 mA for experiments using electrical reducing power and NR.Bacterial cells were cultivated for 16 hours in medium A withfumarate-H₂ or glucose. The cells were anaerobically harvested bycentrifugation at 5,000×g and 20° C. for 30 minutes and washed twicewith 100 mM KCl. The cells were modified with 100 μM NR to measureelectrical-dependent proton translocation and washed again with 100 mM KCl. The washed bacteria (OD₆₆₀, 10) were re-suspended in N₂-saturated150 mM KCl. Cell suspensions were allowed to equilibrate for 30 minutesat room temperature. The incubated cells were centrifuged at 5,000×g and20° C. for 30 minutes and re-suspended in KKG solution and then theincubation was continued for 30 minutes under H₂ atmosphere before themeasurement of proton translocation. To measure electrical-dependentproton translocation upon fumarate addition, the cell suspension wasincubated in the presence or absence of HOQNO in the cathode compartmentunder N₂ atmosphere and charged with 2.0 volt electrode potential for 20minutes.

Enzyme Assays

Enzyme activity measurements were performed under an anaerobic N₂atmosphere, as described previously (Van der Werf, et al., 1997, Arch.Microbiol. 167:332-342). The membrane-free extract, purified membraneand solubilized membrane preparations described above were used to assayhydrogenase, diaphorase, and fumarate reductase activities. Fumaratereductase (EC 1.3.) and hydrogenase (EC 2.12.2.2.) activities weremeasured as described by van der Werf (1997, Arch. Microbiol.167:332-342), with a Beckman spectrophotometer (Model, DU-650).Diaphorase activity with BV2⁺ and NR⁺ was measured under analogousconditions with hydrogenase using NADH (0.6 mM) instead of H₂ aselectron donor (Schneider, et al., 1984, Eur. J. Biochem. 142:75-84).The oxidation and reduction of benzyl viologen and NR werespectrophotometrically measured at 578=m and 540 nm, and the oxidationand reduction of NAD(H) were spectrophotometrically measured at 340 nm.Reduced benzyl viologen was prepared as described previously (Lissolo,et al., 1984, J. Biol. Chem. 259:11725-11729). The millimolar extinctioncoefficient of benzyl viologen (578), NR (540) and NAD(H) (340) were8.65 mM⁻¹ cm⁻¹, 7.12 mM⁻¹ cm⁻¹, and 6.23 mM⁻¹ cm⁻¹, respectively.

Enzymatic Analysis of Fumarate Reduction Membranes and SolubilizedMembrane

Membrane suspensions (3.25 mg/ml protein) and solubilized membranes (3.2mg/ml protein) were used as the enzyme sources. Serum vials (50 ml) andECB system I was used for H₂-dependent and electrical dependentreduction of fumarate to succinate, respectively. Anaerobically prepared50 mM fumarate in 50 mM phosphate buffer (pH 7.2) was used as reactantand catholyte, and 100 mM phosphate buffer with 100 mM NaCl (pH 7.0) wasused as anolyte. The reaction was started by the addition of enzymesources and it was maintained at 37° C. Substrate and productconcentrations were analyzed by HPLC (Guerrant, et al., 1982, J. Clin.Microbiol. 16:355-360). The influence of HOQNO on fumarate reduction incell suspensions and membranes were analyzed as follows.

Cell suspensions (OD₆₆₀=4.2) and membrane suspension (2.65 mg/mlprotein) were used as the enzyme sources. Serum vials (50 ml) and ECBsystem I was used for H₂-dependent and electrical dependent reduction offumarate to succinate, respectively. Anaerobically prepared 50 mMfumarate in 50 mM phosphate buffer (pH 7.2) was used as reactant andcatholyte and 100 mM phosphate buffer with 100 mM NaCl (pH 7.0) was usedas analyte. 2 μM HOQNO was used as an inhibitor for menaquinone. Thereaction was started by the addition of enzyme sources and it wasmaintained at 37° C. Substrate and product concentration was analyzed byHPLC.

Cyclic Voltammetry

A 3 mm diameter glassy carbon working electrode (BAS, West Lafayette,Ind., USA), platinum wire counter electrode (BAS), and an Ag/AgClreference electrode (BAS) were used in an electrochemical cell with aworking volume of 2 ml. Cyclic voltammetry was performed using a cyclicvoltametric potentiostat (BAS, model CV50W) linked to an IBMmicrocomputer data acquisition system. Prior to use, the workingelectrode was polished with an alumina/water slurry on cotton wool, andthe electrochemical cell was thoroughly washed. Oxygen was purged fromthe cell suspension, membrane suspension, or solubilized membranesolution by bubbling with oxygen free N₂ for 10 minutes beforeelectrochemical measurements. Bacterial suspensions (OD₆₆₀=3.0),membrane suspensions (2.54 mg protein/ml), and solubilized membranes(3.2 mg protein/ml) were used as enzyme sources. The scan rate used was25 mV/s over the range −0.3 to −0.8 volt 50 mM phosphate buffercontaining 5 mM NaCl was used as electrolyte. NR μ100 (M) and 50 mMfumarate was used as the electron mediator and the electron acceptor,respectively.

Growth Analysis

Growth of cells suspended in the medium was determined by measuring thesuspensions (optical density at 660 nm), the growth yield of cellsabsorbed onto the electrode was determined by measuring proteinconcentration. The protein concentration was converted to opticaldensity using a predetermined calibration curve (bacterialdensity=protein concentration, mg/ml×1.7556). The cathode, on which thebacteria absorbed, was washed three times, by slow agitation, in 300 mlof phosphate buffer (50 mM, pH 7.0) for 30 minutes. The bacterial lysatewas obtained from electrodes by alkaline treatment at 100° C. for 10minutes using 1N-NaOH. After removing cell debris from the lysate bycentrifugation at 10,000×g and 4° C. for 30 minutes, the proteinconcentration of the bacterial lysate was determined using BradfordReagent (Bio-Rad, Hercules, Calif., USA), and a predeterminedcalibration curve (protein concentration, mg/ml=A₅₉₅×1.3327).

Methanogenic Granules Growth and Metabolic Analysis

Methanogenic granules containing mixed cultures of fatty acid-degradingsyntrophiles and methanogens were obtained from a bench scale anaerobicsludge reactor fed on a mixture of 50 mM acetate, butyrate, andpropionate in MBI International (Lansing, Mich.) (Wu, et al., 1993,Arch. Microbiol. 39:795-803 and Wu, et al., 1993, Appl. Mirobiol.Biotechnol. 39:804-811). Methanogenic granules were cultivated in PBBMprepared without organic compounds (Kenealy, et al., 1981, J. Bacteriol.146:133-140). The medium was prepared without phosphate, brought to pH7.2 with NaOH, boiled, sparged with N₂—CO₂ (80:20%) or H₂—CO₂ (80:20%),dispensed into 158-ml Wheaton serum vials, sealed with butyl rubberstoppers, and autoclaved. Phosphate, sulfide (0.01%), N₂—CO₂ (80:20%) orH₂—CO₂ (80:20%), and vitamin solution were added after autoclaving. Themedium volume was 40 ml, and the initial head space gas pressure inserum vials was adjusted to 30 psi. Media were inoculated with 3.0% (byvolume; protein concentration, 1.995 mg/ml) methanogenic granules andincubated at 37° C. All procedures for medium preparation, inoculation,and cultivation were the same as those used for vial cultures exceptthat Na₂S was not added because the medium was electrically reduced.Na₂S (2%) was added to the anode compartment as reducing agent to removethe O₂ generated. NR (100 (M) was added to the cathode compartment aselectron mediator. The current and potential between anode and cathodewere 0.4 mA and 2.0 volts. CO₂ and CH₄ were analyzed using a gaschromatograph equipped with a carbosphere column and flame ionizeddetector. The injector and column temperatures were 50° C. and 150° C.,respectively, and the carrier (N₂) flow rate was 45 ml/min. Gas sampleswere removed with a pressure lock syringe. CO₂ consumption and CH₄production are shown as the percentage of total gas composition in theheadspace.

Bacterial Growth and Cell Preparation for Generating Electricity

A. succinogenes 130Z and E. coli K-12 were anaerobically grown for 16hours and 20 hours, respectively, in medium A (10 g/L glucose, 5 g/Lyeast extract, 8.5 g/L NaH₂PO₄, and 10 g/L NaHCO₃) under an anaerobicN₂—CO₂ (80:20) atmosphere at 37° C. in 150 ml serum vials or under a N₂(100%) atmosphere in fuel cell system with a pH controller. The inoculumsize was 3% (v/v) for both vial and fuel cell experiments. Resting cellsuspensions were prepared by harvesting stationary phase cultures at 4°C. by centrifugation at 5,000×g. The cells were washed twice using 50 mMphosphate buffer (pH 7.0) under a 100% N₂ atmosphere. The washed cellswere resuspended in 50 mM phosphate buffer (pH 7.0), then dissolved O₂was removed by gassing with N₂ for 30 minutes. The cell density wasadjusted to OD₆₆₀ 3.0.

Fuel Cell Systems for Growing or Resting Cells

A two-compartment (anode and cathode) electrochemical cell was used as afuel cell system for microbial electricity production (FIG. 1). Whenswitches one and two are off, there is an open circuit. When switch oneis on and switch two is off, a closed circuit is formed. When switch oneis off and switch two is on, a closed circuit with external variableresistance is formed. One hundred μM NR or 300 μM thionin were used asthe electron mediator. The total and working volumes of each compartmentwere 1,600 ml and 1,300 ml, respectively. The electrodes, each made of12 g fine woven graphite felt (0.47 m² g, Electrosynthesis, NY) wereconnected to a precision multimeter (Fluke model 45, Everett, Wash.)with a platinum wire ([φ]=0.5 mm, <1.0 Ωcm⁻²; Sigma, St. Louis, Mo.,USA) using graphite epoxy (<1.0 Ωcm⁻², Electrosynthesis, NY). Anode andcathode compartments were separated by a cation-selective membraneseptum ([φ] 70 mm, Nafion, Electrosynthesis, NY). The self-electricresistance of the fuel cell system between the anode and cathode wasapproximately 1,000 Ω. The resistance was adjusted using variableresistance for controlling current production, but it was not adjustedfor measuring maximum potential or current production. The current andvoltage between the anode and cathode were measured by a precisionmultimeter (Fluke model 45, Everett, Wash.). The electrochemicalhalf-reduction of ferric ion (as potassium ferricyanide, E₀(=+0.36volt)—which was re-oxidized by O₂ (E₀′=+0.82 volt) was coupled toneutral red or thionin half-oxidation which was, in turn, reductivelycoupled to bacterial oxidative metabolism. In the fuel cell system usingresting cells, the bacterial cell suspension (OD₆₆₀, 3.0) in 50 mMphosphate buffer (pH 7.2) containing 100 μM NR or 300 μM thionin, and100 mM phosphate buffer (pH 7.0) containing 50 mM ferricyanide were usedas the anolyte and catholyte, respectively. In the fuel cell systemusing growing cells, medium A containing a fresh bacterial inoculum wasthe anolyte; the catholyte was the same as for resting cells. Duringexperiments, complete anoxygenic conditions were maintained in the anodecompartment by gassing with 100% N₂ for 30 minutes before operation atN₂ flow rates of 0.8 ml/min. The trace oxygen contained in the N₂ gaswas removed in a furnace filled with pure copper fillings at 37° C. Thecathode compartment was oxygenated by constant air bubbling andstirring. The anode compartment was maintained at pH 7.0 using anautomatic pH controller (New Brunswick Scientific Co., model pH-40,Edison, N.J.).

Current Production by Chemical Dye Chemical Oxidation Coupled to NADHOxidation

A small chemical fuel cell system (total volume 50 ml; working volume 30ml) consisting of an anode and cathode compartments equipped with 0.3 gfine woven graphite felt electrodes and a cation-selective membraneseptum (Ω 20 mm, Nafion, Electrosynthesis) was used. A 100 μM NRsolution in 50 mM phosphate buffer (pH 7.0) and 100 mM phosphate buffer(pH 7.0) containing 50 mM ferricyanide were used as the anolyte andcatholyte, respectively. Oxygen was completely removed from the anodecompartment by N₂ gassing for 30 minutes before adding NADH. Theconcentrated NADH solution in 50 mM phosphate buffer (pH 7.0) waspreviously gassed with N₂ to remove O₂.

Cyclic Voltametry

A 3 mm-diameter glassy carbon working electrode, a platinum wire counterelectrode, and an Ag/AgCl reference electrode (all from BAS, WestLafayette, Ind.) were used in an electrochemical cell with a 3 mlworking volume. Cyclic voltametry was performed using a cyclicvoltametric potentiostat (model CV50W, BAS) linked to an IBM personalcomputer data acquisition system. Prior to use, the working electrodewas polished with an aluminum/water slurry on cotton wool, and theelectrochemical cell was thoroughly washed. Oxygen was purged from thereactant by bubbling with oxygen-free N₂ for 10 minutes beforeelectrochemical measurement. The scanning rate used was 25 mV/s over therange −0.3 to −0.8 volt. A 50 mM phosphate buffer containing 5 mM NaClwas used as the electrolyte. One hundred μM NR and 100 μM NAD were usedas the electron mediator and acceptor, respectively.

Generation of Electricity Using Anaerobic Sludge

The anaerobic sludge was obtained from the East Lansing sewage treatmentplant (MI, USA). The fresh anaerobic sludge was settled under a N₂atmosphere for one day to remove solid particles. The supernatant (1,200ml) was used as biocatalyst and anolyte for the fuel cell system, towhich 3 g/L glucose was added as energy source. The catholyte was 100 mMphosphate buffer (pH 7.0) containing 50 mM ferricyanide.

Results

Electricity Generation by Fuel Cells

The E₀′ values of the electron mediators used for converting thereducing power generated by microbial metabolic oxidation to electricityare important determinants of the maximum electricity amount that can begenerated in microbial fuel cells. Chemical properties of artificialelectron mediators (i.e., NR and thionin) with those of natural electronmediators (i.e., NAD⁺ and menaquinone) are shown in Table 1. Theelectron driving force generated from using NR is far greater than fromthionin when the redox dye is coupled to an oxidant (i.e., ferricyanide)in a chemical or microbiological fuel cell. This difference is due todifferences between the E₀′ values for NR and thionin. Consequently, theΔE₀ generated from NR or thionin oxidation coupled to ferricyanidereduction is 0.645 volt (NR) and 0.296 volt (thionin). These ΔE₀ valuesare the theoretical maximum potentials produced in fuel cells usingthese electron mediators.

Results of experiments performed demonstrate the superiority of NR overthionin as an electron mediator and that reduced NR is able to donateelectrons to the electrode for electricity production in a microbialfuel cell. FIG. 2 shows that the use of NR as an electron mediator in achemical fuel cell generates higher current than that obtained usingthionin, and that the current produced depends on the NADH concentrationused. Arrows indicate the addition of 1 (circles) or 3.5 (squares) mMNADH. At low NADH concentrations the current was quite low. Althoughthionin reduction was faster than NR reduction when using NADH as thereductant, the mediator oxidation rate at the electrode israte-limiting, because more current was produced with NR as the electronmediator. TABLE 1 Redox mediators, their structural formula, redoxpotentials (E₀ ⁺), and maximum absorbance wavelength (λ_(max).)Structural formula Redox mediator E₀ ⁺(V) λ_(max)

Neutral Red −0.325 540

Thionine +0.064 598

Menaquinone −0.074 260/280

NAD⁺ −0.32 340

Cyclic voltammograms of a NR solution in the presence or absence of NAD⁺show that NR oxidation (upper) and reduction (lower) peaks did not shiftduring twenty scanning cycles in the absence of NAD⁺ (FIG. 3A). Bothpeaks increased upon NAD⁺ addition (FIG. 3B). NAD⁺ enables moreelectrons to pass unidirectionally from the electrode to NR to NAD andfrom NADH to the electrode via NR.

FIG. 4 compares the currents and potentials generated from glucose by E.coli resting cells in a glucose (10 g/L) fuel cell with either 100 μM NR(circles) or 300 μM thionin (squares) in closed circuit (current) (A)and open circuit (potential) (B) configurations. Arrows mark (1) theaddition of the electron mediator; and (2) conversion to open circuit.Under the anaerobic conditions used, higher current and potential levelswere produced with NR than with thionin as the electronophore. Incontrol experiments under aerobic conditions, significant levels ofcurrent, or potential were not detectable because NR and thionin cannotoxidize NADH through the electron transport system since O₂ is a muchbetter electron acceptor (i.e., it has a much more positive E₀′ valuethan the two electron mediators). Under anaerobic conditions, E. colinormally couples NADH oxidation with reduction of either fumarate tosuccinate, acetyl CoA to ethanol, or pyruvate to lactate. Thesereactions are inhibited in the presence of NR in the fuel cell, andelectricity is produced in lieu of these normal reduced metabolic endproducts.

Previous investigations (Allen, et al., 1993, Appl. Biochem. Biotechnol.39-40:27-40 and Thurston, et al., 1985, J. Gen. Microbiol.131:1393-1401) have shown in microbial fuel cells using thionin as theelectron mediator, that both current and potential drop when the restingcells are depleted of glucose. We performed experiments to determinewhat maximal electrical productivities and stabilities can be generatedby resting E. coli cells from different glucose concentrations in a fuelcell with NR as the electronophore. Table 2 shows the effect of glucoseconcentration on the maximal electrical productivities and stabilitiesin an open circuit versus a closed circuit, with and without a 120 ohmexternal resistance. The maximal current, potential, and electricalenergy produced by the fuel cell were proportional to the glucose (i.e.,fuel) concentration. The maximum current and coulombic yields obtainedfrom glucose using NR as the electronophore far exceeded those obtainedwith thionin in other investigations (Dealney, et al., 1984, Chem. Tech.Biotechnol. 34B: 13-27).

Previous studies (Roller, et al., 1984, J. Chem. Tech. Biotechnol.34B:3-12 and Bennetto, et al., 1985, Biotechnol. Lett. 7:699-105) onmicrobial fuel cells with thionin as the electron mediator were onlyperformed with resting cell suspensions (i.e., cells harvested aftergrowth had ended). Using NR (100 uM) as the electronophore, we comparedthe electrical productivities (i.e., current and potential) of A.succinogenes growing cells (FIG. 5A) and resting cells (FIG. 5B) in aglucose (10 g/L) microbial fuel cell under anaerobic conditionswith(FIG. 5). Control experiments (FIG. 5A) showed that the growth yieldand rate (squares) were much higher in the absence of NR when noelectricity was generated (triangles) than in the presence of NR. Theelectric current (open circles) and potential (closed circles) generatedincreased with cell growth. The potentials generated by growing andresting cells were similar, whereas the current produced by restingcells was significantly higher (about 2-fold) than that produced bygrowing cells. The specific current produced per mg cell protein perhour was calculated at 10 hours for growing cells (1.235 mA/mgprotein/hr) and at 2 hours for resting cells (2.595 mA/mg protein/hr)when the glucose levels were high. A total of 68 coulombs was producedby growing cells at 20 hours (after glucose was depleted); whereas theresting cells had produced 90 coulombs at 4 hours. TABLE 2 Effect ofinitial glucose concentration on electrical productivity and stabilityof a microbial fuel cell using E. coli resting cells and NR as theelectronophore. Closed Circuit with a 120Ω Resistance Open CircuitClosed Circuit Electrical Electrical Glucose Potential Current PotentialCurrent Potential Current Coulomb Energy Stability (mM) (volt) (mA)(volt) (mA) (volt) (mA) (amp/sec) (J) (hr) 11.1 0.58 0.0 0.02 1.2 0.460.5 57.6 26.5 32 55.5 0.65 0.0 0.04 5.6 0.57 3.6 1049.76 598.4 81 1110.85 0.0 0.05 17.7 0.62 4.8 2039.04 1264.2 118

Similar experiments were performed using an aerobically grown E. colicells in the presence or absence of electrical generation. Electricalgeneration dramatically decreases growth yield, ATP yield and metaboliteproduction (Table 3). Table 4 compares substrate consumption, growth andelectricity production by exponential versus stationary phase E. colicells. These data indicate that significantly more electricity isproduced by stationary phase cells than by exponential phase cells. Thisresult was expected because significant reducing power is required forcell growth that cannot be directed to electricity generation. TABLE 3Comparison of anaerobic metabolism of E. coli during anaerobic growth inthe presence or absence of electrical generation^(a). Glucose Cell YsubTheoretical ATP Electricity Growth Consumption Mass (g cell/mol Productsyield (mol/mol Energy (J/mol Condition (mM) (g/L) substrate) (mM) sub)sub) Without 60.6 3.07 50.12 11.93 7.07 — Electricity Generation With66.3 1.4088 22.082 8.88 2.57 1320.0 Electricity Generation^(a)Data was determined after 20 hours of growth in medium A with 100 μMneutral red in a standard fuel cell.

TABLE 4 Comparison of substrate consumption and electricity productionby anaerobic E. coli exponential phase versus stationary phase cells ina fuel cell using neutral red as electronophore^(a). Exponential PhaseCells Stationary Phase Cells Electricity Electricity Glucose EnergyGlucose Energy Consumption Cell Mass (J/mol sub) Consumption Cell Mass(J/mol sub) 45.1 mM 1.74 g/L 100.8 15.5 mM 0.214 g/L 1207.7 (7.52 mM/hr)(0.29 g/L/hr) (2.59 mM/hr) (0.035 g/L/hr)^(a)Data for exponential phase cells is from 0-6 hours afterinoculation. Data for stationary phase cells is 12-18 hours afterinoculation. Conditions: medium A with 100 μM neutral red in thestandard fuel cell system.

Experiments were initiated using anaerobic sludge to test its potentialas a catalyst for electricity generation in a fuel cell with NR as theelectronophore. FIG. 6 shows the effect of glucose addition on thecurrent and potential generated by the sewage sludge, as well as themaximum current produced in a closed circuit configuration versus themaximum potential produced in an open circuit configuration. Thenumbered arrows connote the conversion from open to closed circuit witha 2.2 kohms resistance (1); addition of 3 g/L glucose (2); conversionfrom closed to open circuit (3); and the conversion from open to closedcircuit without external resistance (4). The electrical productivity ofthe glucose fuel cell using sewage sludge as the catalyst was calculatedto be a total of 370.8 C (G of 162.82 J).

We have shown here that NR serves as a superior electronophore orelectron mediator than thionin in microbial fuel cells using glucose asfuel. Furthermore, we have shown that resting cells generate moreelectricity than growing cells, and that mixed cultures such as sewagesludge can be robust catalysts for electricity generation in fuel cellsutilizing NR as the electron mediator.

FIG. 7 summarizes our working model explaining E. coli (or A.succinogenes) metabolic properties in fuel cells using NR as theelectron mediator versus during normal (A) versus electrogenic glucosemetabolism (B) of E. coli or A. succinogenes in a fuel cell with NR asthe electronophore. Cell growth, ATP synthesis, and reduced end productformation decrease in relation to the amount of electricity generated.In the presence of NR, cell growth is significantly reduced and NADH isoxidized via NR-mediated electrical generation in lieu of producingnormal reduced end products (i.e., succinate, lactate, and ethanol).Cells still generate ATP by substrate-level phosphorylation (i.e.,acetate kinase) but grow slower because they cannot generate ATP byelectron transport-mediated phosphorylation (i.e., fumarate reductase).

NR is superior to thionin as an electron mediator because it enhancesboth the rate of electron transfer (current) and the yield of electronstransferred (coulombic yield). The highest current (>17 mA) produced ina microbial fuel cell using NR was significantly higher than thatachieved previously with thionin as the electron mediator (Roller, etal., 1984, J. Chem. Tech. Biotechnol. 34B:3-12 and Allen, et al., 1993,Appl. Biochem. Biotechnol. 39-40:27-40); it is, however, still low inelectrical terms. There may be potential applications for low-power DCmicrobial fuel cells such as to maintain telecommunications in remoteareas including outer space.

Example 2 Bioreduction of Ketones to Alcohols

Introduction

Bioreductions of ketones require either NADH or NADPH as a co-factor andthe difficulty in implementing an efficient and economical recyclingsystem has restricted large scale applications to whole cell processes.Specifically, we are interested in asymmetric bioreduction of aβ-tetralone to its corresponding (S)-alcohol by the yeast Trichosporoncapitatum. The alcohol, β-tetralol, is subsequently used in thesynthesis of MK-0499, a very potent potassium channel blocker whichmediates polarization of cardiac tissues.

Recently, we have developed an electrochemical co-factor recyclingtechnology using an electrochemical bioreactor system (described above).The technology was examined and demonstrated in fermentation processesproducing organic acids and in reducing CO₂ to CH₄ with an activatedsludge, resulting in a 20-40% increase in the end product concentration.

We have evaluated the electrochemical co-factor (NAD) recyclingtechnology using either whole cells of Trichosporon capitatum (strain MY1890) or an oxidoreductase isolated from this organism, to support thebioreduction of 6-bromo-β-tetralone (L735,707) to its correspondingalcohol 6-bromo-β-tetralol. In this Example, we present the data onincreased biotransformation rate and β-tetralol concentration using thisnew electrochemical co-factor technology.

Materials and Methods

Electrochemical Bioreactor System

An electrochemical bioreactor (ECB) was designed and constructed toconduct biostransformation in the presence and absence of anelectrically reduced system. The ECB was separated into anode andcathode compartments by a cation selective membrane septum (φ 22 mm,Nafion, Electrosynthesis, NY). The anode and cathode electrodes weremade from graphite fine woven felt (6 mm thickness, 0.47 m²g-1 ofavailable surface area, Electrosynthesis, NY). The weights of theelectrodes were adjusted to 0.5 g (10×50 mm). The voltage and currentbetween the anode and cathode were measured using a precisionmultimeter, and were adjusted to 0.7-10.0 volt and 0.5-10 mA,respectively. The total volume of each compartment was 70 mL with aworking volume of 40 mL. The reaction mixture was placed in the cathodecompartment, whereas 100 mM phosphate buffered saline was filled in theanode compartment.

Microorganism and Medium Composition

Trichosporon capitatum MY 1890 (received from the Merck & Co.) was grownon culture medium containing glycerol, 30 g/L; soytone, 25 g/L and yeastextract, 10 g/L. The culture was grown in 200 mL medium in a 1 L baffledflask and was incubated on a 200 rpm orbital shaker at 29-30° C. for 48hours.

Preparation of Biomass for Biotransformation

The cells from a 48 hour grown culture were harvested by centrifugationat 8000 rpm for 30 minutes. The cell pellet was resuspended in the samevolume (200 ml) of 50 mM Tris buffer (pH 7.0), and washed twice with thebuffer by centrifugation.

Biotransformation with Biomass

The washed cells were resuspended in 50 mM Tris buffer (pH 7.0) in anappropriate volume to achieve 1×, 2× and 3× biomass concentration. Thereaction mixture contained cell suspension, β-bromo-tetralone, ethanol,100 nM neutral red and 50 mM Tris buffer (pH 7.0). Biotransformation wasconducted in the ECB system on a reciprocal shaking incubator (200strokes/min) at 30° C.

Analysis of Substrate and Product

β-Bromo-tetralone and β-bromo-tetranol were analyzed by a Waters HPLC640 equipped with a Zorbax RX-C8 column. The absorbency was measured at220 nm. The mobile phase containing 50% acetonitrile and 50% acidifiedwater (0.1% phosphoric acid) mixture was used at a flow rate of 1.0ml/L.

Results and Discussions

Biotransformation of β-tetralone to β-tetralol with a 48 hourTrichosporon capitatum Culture in the Presence of 1.5 Volt Electricity

Comparison of the biotransformation in the presence of 1.5 volt to theabsence of electricity was carried out with 1 g/L of substrate in theelectrochemical bioreactor system. As shown in FIG. 9, the rate ofβ-tetralol formation in the presence of the electricity wassignificantly higher than without the electricity. The overall reactionrate during the first 3 hour reaction was increased by 45%. Although thereaction rate gradually decreased, the high initial reaction rateresulted in a faster completion of the biotransformation. In thepresence of the electricity the product formation was completed in 3hours. However, in the absence of the electricity, the reaction wasprolonged up to 8 hours and the final end product concentration waslower than what was achieved with the electricity (0.53 g/L). It isimportant to note the high initial reaction rate was observed within thefirst hour in the presence of electricity.

Biotransformation of β-tetralone at 2 g/L of Substrate

Assuming that the biotransformation yield was limited by theavailability of the substrate, the level of substrate was increased to 2g/L while maintaining 1.5 volt of electricity. The product concentrationincreased to 1.2 g/L in about 3-4 hours (FIG. 10). This productconcentration correlates well with 053 g/L at 1 g/L of substrate innearly the same reaction time. The initial reaction rate was alsodramatic in this experiment (2 g/L substrate). Based on these results,it was decided to maintain 2 g/L substrate level to achieve higherlevels of reaction rates and the end product.

Biotransformation with Pulse Feeding of Substrate

To maintain the higher substrate concentration in the reaction mixture,pulse feeding of 2 g/L of substrate was done at 1.5-2 hour interval fora total of 4 times. Although pulse feeding of the substrate was followedby an increase in product formation up to 2.2. g/L in the presence ofelectricity (FIG. 11), this increase was not in proportion to the amountof total fed substrate. Because the substrate β-tetralone has a very lowsolubility in the aqueous phase, a larger part of the substrate remainedin the insoluble form, and some of it was probably also adsorbed ontothe graphite felt of the electrode. Thus, only limited substrate wasconverted to the end product. To reduce the unutilized substrate, theconcentration of the substrate pulse fed to the reaction mixture wasreduced to 1 g/L. The results indicate that although productconcentration decreased from 2.2 g/L (at 2 g/L substrate) to 1.8 g/L at1 g/L substrate (pulse feeding), there was almost no change in thereaction rate when the substrate was pulse fed at 1 g/L (FIG. 12).

Effect of Ethanol Concentration on the Biotransformation

Since the substrate was poorly soluble in water, it was dissolved in 5%ethanol. Assuming that a higher dissolved substrate might result inincreased end-product concentration in the ECB system, ethanolconcentration was increased from 5 to 50% to increase the amount ofsoluble substrate. As the results presented in FIG. 13, the increasedethanol concentration adversely affected the reaction rate and resultedin a low final product formation presumably due to ethanol toxicity. Thehighest reaction rate as well as the product concentration was achievedwith 5% ethanol. Thus, another method has to be applied to increase thesolubility of the substrate to test the hypothesis that a higherdissolved substrate will result in high biotransformation rates andproduct yields.

Effect of Various Levels of Electricity on Biotransformation Rate

Since the biochemical reactions are affected by the amount of availablereducing power, we examined the effect of various levels of electricityon the rate of the biotransformation of β-tetralone to β-tetralol. Inthe first experiment, 0, 0.7, 1.4 and 2.1 volt of electricity wassupplied. In the second experiment, the electricity was increased up to10 volt. For these two experiments, two different batches were used. Asshown in FIGS. 6(1) and 6(2), electricity up to 5 volts enhanced thereaction rate and the final product concentration. It seems that ahigher electric supply provides a higher driving force for the reductivereaction. However, the reaction rate was drastically affected within 20minutes with 10 volts of electricity. It is likely that the highelectrical potential of 10 volts caused the denaturation of protein,thus affected the cellular metabolic reactions. The initial reactionrate with different levels of electricity are presented in Table 5. Theresults demonstrate that the electricity enhanced the reaction rate,e.g. with 5 volts electricity, the biotransformation rate increased totwo-fold in comparison to the rate without the electricity. TABLE 5Relationship between electrical potential and the biotransformation rateReaction rate (mg/L/min) in 20 minutes Voltage 1^(st) trial 2^(nd) trial0 17.0 14 (control) 0.7 21.0 — 1.4 23.0 — 2.1 26.0 21.5 3.5 — 24.5 5.0 —26.9 10.0 — 12.6Biotransformation with Variable Biomass, Voltage and Substrate in aMatrix Design

Our results have demonstrated a high initial rate of biotransformationof β-tetralone to β-tetralol in the ECB system. We have also observed anelectricity-dependent increase in the reaction rate as well as asubstrate concentration-dependent increase in the β-tetralolconcentration. However, these experiments do not explain the cumulativeeffect of the electricity, substrate concentration and the amount ofbiomass on the reaction rate.

Therefore, to examine the combined effect of biomass, electricity andsubstrate on the biotransformation of β-tetralone, we conductedexperiments according to a “BB matrix design 6” provided by Merck & Co.Initial reaction rates were determined within 10 minutes and thesemeasurements were accomplished by using 4 different batches of theculture with internal controls. Comparison of the initial reaction ratesat different substrate concentrations shows that the specific reactionrates were significantly affected by the electricity and biomass athigher substrate concentration (up to 4 g/L), but not at 1 g/L (Table6). Similarly, a higher electricity (6 volt) resulted in a high specificreaction rate (8.1 mg/L/min/g) at a moderate level of biomass and highsubstrate concentration or vice versa. A similar reaction rate was alsoachieved by a moderate level of electricity (3.5 volt), but under highbiomass (3×) and substrate (4 g/L) conditions. Our results also showthat low biomass concentrations resulted in a significantly enhancedspecific reaction rate (6.46 mg/L/min/g) by provide high electricity.However, an increased biomass (2×) was necessary to obtain the similarreaction rate (6.54 mg/L/min/g) if electricity was reduced from 6.0 to3.5 volt (Table 6). TABLE 6 Cumulative effect of biomass, voltage andsubstrate on the initial specific reaction rate in a BB Matrix Design 6.Substrate Initial reaction Specific reaction Biomass Voltage Conc. (g/L)rate (mg/L/min) rate (mg/L/min) Culture batch #1: Dry weight of biomass:10.23 g/L 1.0 3.5 1.0 24.6 2.40 2.0 3.5 2.5 46.7 4.57 2.0 1.0 4.0 56.45.52 3.0 3.5 2.5 66.9 6.54 0.0 0.0 2.5 0.0 0 1.0 3.5 4.0 48.3 4.72 1.01.0 2.5 29.8 2.91 2.0 0.0 2.5 31.2 3.05 Culture batch #2: Dry weight ofbiomass: 7.31 g/L 1.0 3.5 4.0 59.6 8.15 1.0 6.0 2.5 47.2 6.46 2.0 3.52.5 32.8 4.49 2.0 6.0 4.0 59.7 8.17 Culture batch #3: Dry weight ofbiomass: 7.21 g/L 3.0 1.0 2.5 44.0 6.10 2.0 1.0 1.0 20.2 2.80 3.0 6.02.5 58.7 8.14 2.0 6.0 1.0 26.8 3.72 Culture batch #4: Dry weight ofbiomass: 8.20 g/L 3.0 3.5 1.0 33.5 4.08 2.0 6.0 4.0 73.4 7.98 2.0 3.52.5 37.4 4.56 1.0 0.0 2.5 19.5 2.38Biotransformation with Purified β-tetralone Reductase

Enzymatic biotransformation was conducted with 0.02 unit/ml of thepurified β-tetralone reductase, 10 mM NAD and 10 mM β-tetralone in thepresence of 0, 1.0 and 3.0 volt of electricity. To confirm the enzymereaction, one reaction mixture was prepared as a control with NADH₂instead of NAD, in the absence of electricity. The results show that0.13 g/L of the product was produced with 3 volt of electricity after 8hour incubation, whereas only a trace amount of the product was found inthe reaction mixture with 0 and 1.5 volt of electricity (FIG. 14). Incomparison, about 0.8 g/L of the product was produced with NADH₂. It isnot clear why the enzymatic reaction rate with 3 volt electricity wasslow.

CONCLUSION

In this study, it was demonstrated that the initial rate ofbiotransformation of β-tetralone to β-tetralol and the final productconcentration were enhanced due to electricity-based reducing power inthe electrochemical bioreactor system. It was found that thebiotransformation rate was significantly affected by the amount ofbiomass, substrate concentration and electrical potential. The influenceof electrical potential on the biotransformation rates was moresignificantly observed at a high substrate level of 2.5-4 g/L. The ECBsystem has shown a great potential in reducing the reaction tine that islikely to result in significant cost savings.

The present invention is not limited to the exemplified embodiments, butis intended to encompass all such modifications and variations as comewithin the scope of the following claims.

1. A method of promoting reductive processes in a biological systemcomprising the steps of: (a) providing an electrochemical bioreactorsystem having a cathode compartment equipped with a cathode and an anodecompartment equipped with an anode, the cathode and anode compartmentbeing separated by a cation selective membrane, wherein the cathode andanode are connected by a conductive material to a power supply; (b)placing a suitable amount of neutral red, a biological catalystincluding Trichosporon capitatum, and a substrate in the cathodecompartment; and (c) applying from the power supply a current betweenthe anode and the cathode, wherein at least a portion of the neutral redis electrically reduced and the substrate is converted to an alcohol.